Glycocalyx Curving the Membrane: Forces Emerging from the Cell Exterior.

Morphological transitions are typically attributed to the actions of proteins and lipids. Largely overlooked in membrane shape regulation is the glycocalyx, a pericellular membrane coat that resides on all cells in the human body. Comprised of complex sugar polymers known as glycans as well as glycosylated lipids and proteins, the glycocalyx is ideally positioned to impart forces on the plasma membrane. Large, unstructured polysaccharides and glycoproteins in the glycocalyx can generate crowding pressures strong enough to induce membrane curvature. Stress may also originate from glycan chains that convey curvature preference on asymmetrically distributed lipids, which are exploited by binding factors and infectious agents to induce morphological changes. Through such forces, the glycocalyx can have profound effects on the biogenesis of functional cell surface structures as well as the secretion of extracellular vesicles. In this review, we discuss recent evidence and examples of these mechanisms in normal health and disease.

Physical Principles of Membrane Shape Regulation by the Glycocalyx.

Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix.

Immunoengineering can overcome the glycocalyx armour of cancer cells.

Cancer cell glycocalyx is a major line of defence against immune surveillance. However, how specific physical properties of the glycocalyx are regulated on a molecular level, contribute to immune evasion and may be overcome through immunoengineering must be resolved. Here we report how cancer-associated mucins and their glycosylation contribute to the nanoscale material thickness of the glycocalyx and consequently modulate the functional interactions with cytotoxic immune cells. Natural-killer-cell-mediated cytotoxicity is inversely correlated with the glycocalyx thickness of the target cells. Changes in glycocalyx thickness of approximately 10 nm can alter the susceptibility to immune cell attack. Enhanced stimulation of natural killer and T cells through equipment with chimeric antigen receptors can improve the cytotoxicity against mucin-bearing target cells. Alternatively, cytotoxicity can be enhanced through engineering effector cells to display glycocalyx-editing enzymes, including mucinases and sialidases. Together, our results motivate the development of immunoengineering strategies that overcome the glycocalyx armour of cancer cells.

Balancing forces: architectural control of mechanotransduction.

All cells exist within the context of a three-dimensional microenvironment in which they are exposed to mechanical and physical cues. These cues can be disrupted through perturbations to mechanotransduction, from the nanoscale-level to the tissue-level, which compromises tensional homeostasis to promote pathologies such as cardiovascular disease and cancer. The mechanisms of such perturbations suggest that a complex interplay exists between the extracellular microenvironment and cellular function. Furthermore, sustained disruptions in tensional homeostasis can be caused by alterations in the extracellular matrix, allowing it to serve as a mechanically based memory-storage device that can perpetuate a disease or restore normal tissue behaviour.

Investigation of synovial fluid lubricants and inflammatory cytokines in the horse: a comparison of recombinant equine interleukin 1 beta-induced synovitis and joint lavage models.

BACKGROUND: Lameness is a debilitating condition in equine athletes that leads to more performance limitation and loss of use than any other medical condition. There are a limited number of non-terminal experimental models that can be used to study early inflammatory and synovial fluid biophysical changes that occur in the equine joint. Here, we compare the well-established carpal IL-1ß-induced synovitis model to a tarsal intra-articular lavage model, focusing on serial changes in synovial fluid inflammatory cytokines/chemokines and the synovial fluid lubricating molecules lubricin/proteoglycan 4 and hyaluronic acid. The objectives of this study were to evaluate clinical signs; synovial membrane and synovial fluid inflammation; and synovial fluid lubricants and biophysical properties in response to carpal IL-1ß synovitis and tarsal intra-articular lavage. RESULTS: Hyaluronic acid (HA) concentrations, especially high molecular weight HA, and synovial fluid viscosity decreased after both synovitis and lavage interventions. Synovial fluid lubricin concentrations increased 17-20-fold for both synovitis and lavage models, with similar changes in both affected and contralateral joints, suggesting that repeated arthrocentesis alone resulted in elevated synovial fluid lubricin concentrations. Synovitis resulted in a more severe inflammatory response based on clinical signs (temperature, heart rate, respiratory rate, lameness and joint effusion) and clinicopathological and biochemical parameters (white blood cell count, total protein, prostaglandin E2, sulfated glycosaminoglycans, tumor necrosis factor-α and CC chemokine ligands - 2, - 3, - 5 and - 11) as compared to lavage. CONCLUSIONS: Synovial fluid lubricin increased in response to IL-1ß synovitis and joint lavage but also as a result of repeated arthrocentesis. Frequent repeated arthrocentesis is associated with inflammatory changes, including increased sulfated glycosaminoglycan concentrations and decreased hyaluronic acid concentrations. Synovitis results in more significant inflammatory changes than joint lavage. Our data suggests that synovial fluid lubricin, TNF-α, CCL2, CCL3, CCL5, CCL11 and sGAG may be useful biomarkers for synovitis and post-lavage joint inflammation. Caution should be exercised when performing repeated arthrocentesis clinically or in experimental studies due to the inflammatory response and loss of HA and synovial fluid viscosity.

Direct comparison of optical and electron microscopy methods for structural characterization of extracellular vesicles.

As interest in the role of extracellular vesicles in cell-to-cell communication has increased, so has the use of microscopy and analytical techniques to assess their formation, release, and morphology. In this study, we evaluate scanning electron microscopy (SEM) and cryo-SEM for characterizing the formation and shedding of vesicles from human breast cell lines, parental and hyaluronan synthase 3-(HAS3)-overexpressing MCF10A cells, grown directly on transmission electron microscopy (TEM) grids. While cells imaged with conventional and cryo-SEM exhibit distinct morphologies due to the sample preparation process for each technique, tubular structures protruding from the cell surfaces were observed with both approaches. For HAS3-MCF10A cells, vesicles were present along the length of membrane protrusions. Once completely shed from the cells, extracellular vesicles were characterized using nanoparticle tracking analysis (NTA) and cryo-TEM. The size distributions obtained by each technique were different not only in the range of vesicles analyzed, but also in the relative proportion of smaller-to-larger vesicles. These differences are attributed to the presence of biological debris in the media, which is difficult to differentiate from vesicles in NTA. Furthermore, we demonstrate that cryo-TEM can be used to distinguish between vesicles based on their respective surface structures, thereby providing a path to differentiating vesicle subpopulations and identifying their size distributions. Our study emphasizes the necessity of pairing several techniques to characterize extracellular vesicles.

The cancer glycocalyx mechanically primes integrin-mediated growth and survival.

Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function.

Equilibrium Modeling of the Mechanics and Structure of the Cancer Glycocalyx.

The glycocalyx is a thick coat of proteins and carbohydrates on the outer surface of all eukaryotic cells. Overproduction of large, flexible or rod-like biopolymers, including hyaluronic acid and mucins, in the glycocalyx strongly correlates with the aggression of many cancer types. However, theoretical frameworks to predict the effects of these changes on cancer cell adhesion and other biophysical processes remain limited. Here, we propose a detailed modeling framework for the glycocalyx incorporating important physical effects of biopolymer flexibility, excluded volume, counterion mobility, and coupled membrane deformations. Because mucin and hyaluronic biopolymers are proposed to extend and rigidify depending on the extent of their decoration with side chains, we propose and consider two limiting cases for the structural elements of the glycocalyx: stiff beams and flexible chains. Simulations predict the mechanical response of the glycocalyx to compressive loads, which are imposed on cells residing in the highly confined spaces of the solid tumor or invaded tissues. Notably, the shape of the mechanical response transitions from hyperbolic to sigmoidal for more rod-like glycocalyx elements. These mechanical responses, along with the corresponding equilibrium protein organizations and membrane topographies, are summarized to aid in hypothesis generation and the evaluation of future experimental measurements. Overall, the modeling framework developed provides a theoretical basis for understanding the physical biology of the glycocalyx in human health.

Mucin-coating technologies for protection and reduced aggregation of cellular production systems.

Optimization of host-cell production systems with improved yield and production reliability is desired to meet the increasing demand for biologics with complex posttranslational modifications. Aggregation of suspension-adapted mammalian cells remains a significant problem that can limit the cellular density and per volume yield of bioreactors. Here, we propose a genetically encoded technology that directs the synthesis of antiadhesive and protective coatings on the cellular surface. Inspired by the natural ability of mucin glycoproteins to resist cellular adhesion and hydrate and protect cell and tissue surfaces, we genetically encode new cell-surface coatings through the fusion of engineered mucin domains to synthetic transmembrane anchors. Combined with appropriate expression systems, the mucin-coating technology directs the assembly of thick, highly hydrated barriers to strongly mitigate cell aggregation and protect cells in suspension against fluid shear stresses. The coating technology is demonstrated on suspension-adapted human 293-F cells, which resist clumping even in media formulations that otherwise would induce extreme cell aggregation and show improved performance over a commercially available anticlumping agent. The stable biopolymer coatings do not show deleterious effects on cell proliferation rate, efficiency of transient transfection with complementary DNAs, or recombinant protein expression. Overall, our mucin-coating technology and engineered cell lines have the potential to improve the single-cell growth and viability of suspended cells in bioreactors.

Stable recombinant production of codon-scrambled lubricin and mucin in human cells.

Widespread therapeutic and commercial interest in recombinant mucin technology has emerged due to the unique ability of mucin glycoproteins to hydrate, protect, and lubricate biological surfaces. However, recombinant production of the large, highly repetitive domains that are characteristic of mucins remains a challenge in biomanufacturing likely due, at least in part, to the inherent instability of DNA repeats in the cellular genome. To overcome this challenge, we exploit codon redundancy to encode desired mucin polypeptides with minimal nucleotide repetition. The codon-scrambling strategy was applied to generate synonymous genes, or "synDNAs," for two mucins of commercial interest: lubricin and mucin 1. Stable, long-term recombinant production in suspension-adapted human 293-F cells was demonstrated for the synonymous lubricin complementary DNA (cDNA), which we refer to as SynLubricin. Under optimal conditions, a 293-F subpopulation produced recombinant SynLubricin at more than 200 mg/L of media and was stable throughout 2 months of continuous culture. Functionality tests confirmed that the recombinant lubricin could effectively inhibit cell adhesion and lubricate cartilage explants. Together, our work provides a viable workflow for cDNA design and stable mucin production in mammalian host production systems.

Scanning angle interference microscopy reveals cell dynamics at the nanoscale.

Emerging questions in cell biology necessitate nanoscale imaging in live cells. Here we present scanning angle interference microscopy, which is capable of localizing fluorescent objects with nanoscale precision along the optical axis in motile cellular structures. We use this approach to resolve nanotopographical features of the cell membrane and cytoskeleton as well as the temporal evolution, three-dimensional architecture and nanoscale dynamics of focal adhesion complexes.

Recombinant Production of Glycoengineered Mucins in HEK293-F Cells.

Recombinant mucins are attractive polymeric building blocks for new biomaterials, biolubricants, and therapeutics. Advances in glycoengineered host cell systems now enable the recombinant production of mucins with tailored O-glycan side chains, offering new opportunities to tune the functionality of mucins and investigate the biology of specific O-glycan structures. Here, we provide a protocol for the scalable production of glycoengineered mucins and mucin-like glycoproteins in suspension-adapted HEK293-F cells. The protocol includes the preparation of engineered cell lines with homozygous knockout (KO) of glycosyltransferases using CRISPR/Cas9 and homology-directed repair (HDR) templates designed for efficient screening of clones. Strategies are provided for the stable introduction of mucin expression cassettes into the HEK293-F genome and the subsequent isolation of high-expressing cell populations. The high-titer production of recombinant mucins in conventional shaker flasks is described as an example production strategy using these cell lines.

COLLAGEN MINERALIZATION DECREASES NK CELL-MEDIATED CYTOTOXICITY OF BREAST CANCER CELLS VIA INCREASED GLYCOCALYX THICKNESS.

Skeletal metastasis is common in patients with advanced breast cancer, and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow, but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions. Here, we have utilized a combination of synthetic bone matrix models with controlled mineral content, nanoscale optical imaging, and flow cytometry to evaluate how collagen type I mineralization affects the biochemical and biophysical properties of the tumor cell glycocalyx, a dense layer of glycosylated proteins and lipids decorating their cell surface. Our results suggest that collagen mineralization upregulates mucin-type O-glycosylation and sialylation by tumor cells, which increased their glycocalyx thickness while enhancing resistance to attack by Natural Killer (NK) cells. These changes were functionally linked as treatment with a sialylation inhibitor decreased mineralization-dependent glycocalyx thickness and made tumor cells more susceptible to NK cell attack. Together, our results suggest that interference with glycocalyx sialylation may represent a therapeutic strategy to enhance cancer immunotherapies targeting bone-metastatic breast cancer.

Dimerization activates the Inversin complex in C. elegans.

Genetic, colocalization, and biochemical studies suggest that the ankyrin repeat-containing proteins Inversin (INVS) and ANKS6 function with the NEK8 kinase to control tissue patterning and maintain organ physiology. It is unknown whether these three proteins assemble into a static "Inversin complex" or one that adopts multiple bioactive forms. Through the characterization of hyperactive alleles in C. elegans, we discovered that the Inversin complex is activated by dimerization. Genome engineering of an RFP tag onto the nematode homologues of INVS (MLT-4) and NEK8 (NEKL-2) induced a gain-of-function, cyst-like phenotype that was suppressed by monomerization of the fluorescent tag. Stimulated dimerization of MLT-4 or NEKL-2 using optogenetics was sufficient to recapitulate the phenotype of a constitutively active Inversin complex. Further, dimerization of NEKL-2 bypassed a lethal MLT-4 mutant, demonstrating that the dimeric form is required for function. We propose that dynamic switching between at least two functionally distinct states - an active dimer and an inactive monomer - gates the output of the Inversin complex.

Dimerization activates the Inversin complex in C. elegans.

Genetic, colocalization, and biochemical studies suggest that the ankyrin repeat-containing proteins Inversin (INVS) and ANKS6 function with the NEK8 kinase to control tissue patterning and maintain organ physiology. It is unknown whether these three proteins assemble into a static "Inversin complex" or one that adopts multiple bioactive forms. Through characterization of hyperactive alleles in C. elegans , we discovered that the Inversin complex is activated by dimerization. Genome engineering of an RFP tag onto the nematode homologs of INVS (MLT-4) and NEK8 (NEKL-2) induced a gain-of-function, cyst-like phenotype that was suppressed by monomerization of the fluorescent tag. Stimulated dimerization of MLT-4 or NEKL-2 using optogenetics was sufficient to recapitulate the phenotype of a constitutively active Inversin complex. Further, dimerization of NEKL-2 bypassed a lethal MLT-4 mutant, demonstrating that the dimeric form is required for function. We propose that dynamic switching between at least two functionally distinct states-an active dimer and an inactive monomer-gates the output of the Inversin complex.

Collagen Mineralization Decreases NK Cell-Mediated Cytotoxicity of Breast Cancer Cells via Increased Glycocalyx Thickness.

Skeletal metastasis is common in patients with advanced breast cancer and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions. Here, a combination of synthetic bone matrix models with controlled mineral content, nanoscale optical imaging, and flow cytometry are utilized to evaluate how collagen type I mineralization affects the biochemical and biophysical properties of the tumor cell glycocalyx, a dense layer of glycosylated proteins and lipids decorating their cell surface. These results suggest that collagen mineralization upregulates mucin-type O-glycosylation and sialylation by tumor cells, which increases their glycocalyx thickness while enhancing resistance to attack by natural killer (NK) cells. These changes are functionally linked as treatment with a sialylation inhibitor decreased mineralization-dependent glycocalyx thickness and made tumor cells more susceptible to NK cell attack. Together, these results suggest that interference with glycocalyx sialylation may represent a therapeutic strategy to enhance cancer immunotherapies targeting bone-metastatic breast cancer.

Specific Degradation of the Mucin Domain of Lubricin in Synovial Fluid Impairs Cartilage Lubrication.

Progressive cartilage degradation, synovial inflammation, and joint lubrication dysfunction are key markers of osteoarthritis. The composition of synovial fluid (SF) is altered in OA, with changes to both hyaluronic acid and lubricin, the primary lubricating molecules in SF. Lubricin's distinct bottlebrush mucin domain has been speculated to contribute to its lubricating ability, but the relationship between its structure and mechanical function in SF is not well understood. Here, we demonstrate the application of a novel mucinase (StcE) to selectively degrade lubricin's mucin domain in SF to measure its impact on joint lubrication and friction. Notably, StcE effectively degraded the lubricating ability of SF in a dose-dependent manner starting at nanogram concentrations (1-3.2 ng/mL). Further, the highest StcE doses effectively degraded lubrication to levels on par with trypsin, suggesting that cleavage at the mucin domain of lubricin is sufficient to completely inhibit the lubrication mechanism of the collective protein component in SF. These findings demonstrate the value of mucin-specific experimental approaches to characterize the lubricating properties of SF and reveal key trends in joint lubrication that help us better understand cartilage function in lubrication-deficient joints.

Recombinant manufacturing of multispecies biolubricants.

Lubricin, a lubricating glycoprotein abundant in synovial fluid, forms a low-friction brush polymer interface in tissues exposed to sliding motion including joints, tendon sheaths, and the surface of the eye. Despite its therapeutic potential in diseases such as osteoarthritis and dry eye disease, there are few sources available. Through rational design, we developed a series of recombinant lubricin analogs that utilize the species-specific tissue-binding domains at the N- and C-termini to increase biocompatibility while replacing the central mucin domain with an engineered variant that retains the lubricating properties of native lubricin. In this study, we demonstrate the tissue binding capacity of our engineered lubricin product and its retention in the joint space of rats. Next, we present a new bioprocess chain that utilizes a human-derived cell line to produce O-glycosylation consistent with that of native lubricin and a purification strategy that capitalizes on the positively charged, hydrophobic N- and C-terminal domains. The bioprocess chain is demonstrated at 10 L scale in industry-standard equipment utilizing commonly available ion exchange, hydrophobic interaction and size exclusion chromatography resins. Finally, we confirmed the purity and lubricating properties of the recombinant biolubricant. The biomolecular engineering and bioprocessing strategies presented here are an effective means of lubricin production and could have broad applications to the study of mucins in general.

Bio-orthogonal Glycan Imaging of Culture Cells and Whole Animal C. elegans with Expansion Microscopy.

Complex carbohydrates called glycans play crucial roles in the regulation of cell and tissue physiology, but how glycans map to nanoscale anatomical features must still be resolved. Here, we present the first nanoscale map of mucin-type O -glycans throughout the entirety of the Caenorhabditis elegans model organism. We construct a library of multifunctional linkers to probe and anchor metabolically labelled glycans in expansion microscopy (ExM), an imaging modality that overcomes the diffraction limit of conventional optical microscopes through the physical expansion of samples embedded in a polyelectrolyte gel matrix. A flexible strategy is demonstrated for the chemical synthesis of linkers with a broad inventory of bio-orthogonal functional groups, fluorophores, anchorage chemistries, and linker arms. Employing C. elegans as a test bed, we resolve metabolically labelled O -glycans on the gut microvilli and other nanoscale anatomical features using our ExM reagents and optimized protocols. We use transmission electron microscopy images of C. elegans nano-anatomy as ground truth data to validate the fidelity and isotropy of gel expansion. We construct whole organism maps of C. elegans O -glycosylation in the first larval stage and identify O -glycan "hotspots" in unexpected anatomical locations, including the body wall furrows. Beyond C. elegans , we provide validated ExM protocols for nanoscale imaging of metabolically labelled glycans on cultured mammalian cells. Together, our results suggest the broad applicability of the multifunctional reagents for imaging glycans and other metabolically labelled biomolecules at enhanced resolutions with ExM.

Tensional homeostasis and the malignant phenotype.

Tumors are stiffer than normal tissue, and tumors have altered integrins. Because integrins are mechanotransducers that regulate cell fate, we asked whether tissue stiffness could promote malignant behavior by modulating integrins. We found that tumors are rigid because they have a stiff stroma and elevated Rho-dependent cytoskeletal tension that drives focal adhesions, disrupts adherens junctions, perturbs tissue polarity, enhances growth, and hinders lumen formation. Matrix stiffness perturbs epithelial morphogenesis by clustering integrins to enhance ERK activation and increase ROCK-generated contractility and focal adhesions. Contractile, EGF-transformed epithelia with elevated ERK and Rho activity could be phenotypically reverted to tissues lacking focal adhesions if Rho-generated contractility or ERK activity was decreased. Thus, ERK and Rho constitute part of an integrated mechanoregulatory circuit linking matrix stiffness to cytoskeletal tension through integrins to regulate tissue phenotype.